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Figure 2015.09B. Chromatogram of vitamin K 1

standard.

20 s or stir each sample for at least 20 s at a spin rate that causes a vortex to form within the sample. ( 5 ) Centrifuge the samples until a clean separation of the iso- octane and laboratory water–methanol layers results. The iso- octane layer should be a clear layer at the top of the centrifuge tube, and the laboratory water–methanol layer should be a cloudy layer below the iso-octane layer. (A good separation of solvent layers can usually be achieved by centrifuging samples for approximately 10 min at 800 relative centrifugal force.) ( 6 ) Remove samples from the centrifuge and inspect the samples to verify that the iso-octane and laboratory water–methanol layers are separated. With a glass pipet, carefully rinse down the upper walls of the centrifuge tube with a portion of the iso-octane layer. If the layers become mixed together, centrifuge the sample again. Pipette a portion of the clear iso-octane layer into a labeled autosampler vial and cap the vial. (b)  HPLC analysis . ( 1 )  Instrumental operating conditions .—( a ) HPLC analytical column pump flow rate, 0.4 mL/min. ( b ) Postcolumn flow rate, 0.4 mL/min. ( c ) Injection volume, 20 µL. ( d ) Run time, 20 min. ( e ) Fluorescence excitation and emission, 245 and 440 nm, respectively.

( 2 )  Instrument startup .—The system should be configured as shown in Figure  2015.09B . If necessary, remove used zinc and repack the postcolumn reactor column with fresh zinc. The zinc reactor column should be repacked whenever the S/N in the lowest standard is too high to accurately integrate the vitamin K 1 peak, when peak responses from injections of the same standard drop by more than 7% and the drop cannot be attributed to other system components, or when the system back pressure through the zinc reactor increases significantly and vitamin K 1 peak widths begin to increase. To repack the zinc reactor column, remove the hex nuts and retainers from both ends of the column and force the used zinc out of the column with a thin wire or similar apparatus. Flush the zinc reactor column with ethanol to remove residual zinc. Replace the hex nut and retainer on one end of the zinc reactor column. Carefully transfer a small amount of zinc powder to the reactor column with a spatula, and press down on the zinc in the column with an old HPLC piston or similar apparatus to pack it tightly. Continue adding zinc and pressing it down until the level of zinc is even with the top of the column. After the reactor column is full, replace the second retainer and hex nut. The more tightly zinc is packed into the reactor column, the more symmetrical the vitamin K 1 peaks

Figure 2015.09C. Chromatogram of SRM 1849a.